Introduction:
Antiviral natural products have shown promising alternative for conventional drug-resistance in hepatitis B virus (HBV).

Material and methods:
We evaluated the in vitro hepatocytotoxicity (HepG2 cells; MTT assay) followed by anti-HBV efficacy (HepG2.2.15 cell; HBV-antigens ELISA) of Ilex paraguariensis leaves total-ethanol extract (IP-Ext) and fractionated preparations in n-hexane (IP-Hex), chloroform (IP-Chl), ethyl acetate (IP-EtAc) and ethanol (IP-EtOH).

Results:
All tested samples showed non-cytotoxicity except IP-EtAc having mild toxic effect at 200 μg/ml. Their anti-HBV assessment showed dose-dependent inhibitions of HBV antigens (HBs/HBe). At the selected optimal dose (50 μg/ml), while IP-Ext showed mild (HBsAg: 24.2% & HBeAg: 20.6%) and IP-Chl showed moderate (HBsAg: 42.3% & 40.1%) activities, IP-Hex (HBsAg: 55.6% & HBeAg: 52.4%) and IP-EtOH (HBsAg: 53.2% & HBeAg: 50.2%) exhibited high activities. High-performance liquid chromatography (HPLC) validation identified known anti-HBV flavonoids (Rutin: 18.98, Quercetin: 6.52 and Kaempferol: 9.10 μg/g) and polyphenols (Caffeic acid: 11.43 and Chlorogenic acid: 3.22 μg/g) in the extract. Their estimated anti-HBV activities at 10 μg/ml dose were: Quercetin (HBsAg: 67.8 % & HBeAg: 64.4%), Kaempferol (HBsAg: 63.5 % & HBeAg: 61.6%), Chlorogenic acid (HBsAg: 55.2% & HBeAg: 53.8%), Rutin (HBsAg: 51.2% & HBeAg: 48.4%) and Caffeic acid (HBsAg: 42.2% & HBeAg: 39.5%). Notably, while our previous molecular docking studies had shown strong interactions of these flavonoids with HBV polymerase active-residues, here, we demonstrated good binding-affinities of the polyphenols with drug-sensitive (wild-type) and drug-resistant (mutant) polymerases.

Conclusions:
Taken together, this is the first study suggesting the anti-HBV therapeutic efficacy of I. paraguariensis attributed to its antiviral phytochemicals.